Bangladesh J. Zool. 48(2): 379-391, 2020 ISSN: 0304-9027 eISSN: 2408-8455
TOXICOLOGICAL EFFECT AND BEHAVIORAL RESPONSE OF A PREDATORY STINGING CATFISH, HETEROPNEUSTES FOSSILIS EXPOSED TO THREE INDIGENOUS PLANT SEED EXTRACTS
Sadia Sultana1, Munira Nasiruddin2, M. A. Azadi3*, and M. R. Chowdhury2
Department of Zoology, Noakhali Science and Technology University Noakhali, Bangladesh
Abstract: For the removal of undesired fish species from aquaculture ponds, an alternative to synthetic toxins is the use of botanical toxicants which are biodegradable and environmentally safer. Toxicological effects of distilled water, 50% ethyl alcohol, methanol and acetone extracts of three indigenous plant seeds, Jatropha carcus (Linn.), Hydnocarpus wightianus (Blume) and Aleurites moluccana (Linn. Willd) were studied on the predatory fish, Heteropneustes fossilis (Bloch) under normal laboratory conditions. Depending upon the type of plants, extracts and concentrations, percentage mortality varied. Behavioral activities increased with increasing concentrations. During exposure, fish exhibited discolouration, gulping for air, erratic swimming, loss of reflex, slow movement and ultimately became motionless before death . The extent of toxicity of extracts for J. carcus, H. wightianus and A. moluccana on H. fossilis could be ranked in the order: Acetone > methanol > 50% ethyl alcohol > distilled water. On the basis of
LC50 values the most toxic was acetone extract of J. carcus seeds and least toxic was distilled water extract of A. moluccana seeds. It is suggested that these plant seed extracts would be helpful in aquaculture to remove unwanted fish species from culture ponds.
Key words: Toxicological effect, Heteropneustes fossilis, plant extracts, mortality, lethal concentrations.
INTRODUCTION Predatory fish are those that prey upon other fish or aquatic animals. Frequently introduced, these fish can ruin a pond or lake environment. Problems associated with the stocking of undesirable fishes may include under
*Author for corresponding:
380 Sultana et al.
population of desirable fish species, habitat destruction, competition for food and nesting space, and the small size attained by the introduced fish. The use of piscicide as a tool in pond management during pond preparation to get rid of predators before fish stocking is important. Ideally, ponds should be sundried and the pond bottom crack dried to help to get rid of fish predators. Moreover, farmers who are always in a hurry to prepare their ponds always resort to the use of inorganic/chemical fish toxicants which are deleterious for the environment. Derris root, tea seed cake, camellia seed cake and powdered croton seeds are used as botanical piscicides. The toxic parts of plants employed as fish poisons can include roots, seeds, fruits, bark, latex or leaves (Nasiruddin et al. 2006, 2009a, 2010a, 2012a). The piscicidal potential and phytotoxic properties of different parts of plant extracts have been reported by several researchers namely Ayotunde and Benedict (2008), Ayoola (2011), Akinduyite and Oyedapo (2011), Ayoola et al. (2011), Ayotunde et al. (2011), Fafioye (2012), Kumar et al. (2012), Adesina et al. (2013), Okey et al. (2013), Orji et al. (2014), Oshimagye et al. (2014), Ablaka and Auta (2015), Kishore et al. (2016) and Suely et al. (2016). In Bangladesh, Latifa et al. (2002, 2004), Latifa and Parvin (2006), Nasiruddin and Sultana (2007), Nasiruddin et al. (2006, 2009a, b, 2010a, b, 2011, 2012a, b, 2014), Ara et al. (2012) and Chowdhury et al. (2014) have reported the toxic effect of different plant part extracts on such fish. The test organism is an Asian stinging catfish, Heteropneustes fossilis, is a species of air breathing catfish. It is found mainly in ponds, ditches, swamps, and marshes, but sometimes occurs in muddy rivers. It can tolerate slightly brackish water habitat. It is omnivorous. This species breeds in confined waters during the monsoon months, but can breed in ponds, derelict ponds, and ditches when sufficient rain water accumulates. It has a great demand due to its medicinal value. To overcome the hazardous effects of organic pesticides, recent emphasis is on the use of natural pesticides, which are usually of plant origin. Some plants contain compounds of various classes that have insecticidal and piscicidal properties (Wang and Huffmann 1991). Unlike synthetic chemical pesticides, which leave harmful residues in the aquatic environment (Koesomadinata 1980), botanical pesticides are said to be more environmentally friendly because they are easily biodegraded and leave no residues in the environment. In view of these considerations, the present study was undertaken to study the toxicological effect of three plant seed extracts, Jatropha carcus, Hydnocarpus wightianus and Aleurites moluccana on predatory fish, Heteropneustes fossilis. Their behavioral response was also observed in the present study during Toxicological effect three indigenous plant seed extracts 381 bioassay to assess the toxicity of plant seed extracts. The aim was to develop an effective anesthetic from indigenous plant material that will be available at low cost to aqua culturists and which would be non-toxic to the fish and consumers.
MATERIAL AND METHODS Sample collection and preparation: The toxicants were obtained from the dry seeds (Fig.1) of the plant Jatropha carcus (Family: Euphorbiaceae), Hydnocarpus wightianus (Family: Achariaceae) and Aleurites moluccana (Family: Euphorbiaceae) to carry out the experimental study. These seeds collected from the local area and local seed markets of Chittagong, K.C. Dey Road (22.337965, 91.835605) and Narsingdi Big Bazar (22.4780772, 91.7846100) were cleaned and dried under diffused sunlight for 10-15 days prior to stocking. The seeds were kept in airtight glass bottles in a cool place in the laboratory, so that these seeds remained dry and could be easily grinded for making extracts for experimental purpose.
Seeds of Jatropha carcus Seeds of Hydnocarpus wightianus Seeds of Aleurites moluccana Fig. 1: Dry seeds of Jatropha carcus, Hydnocarpus wightianus and Aleurites moluccana.
The experimental healthy and live fish, Heteropneustes fossilis were collected from local fish markets of Chittagong city on the day of the experiment. Then they were acclimatized in a glass aquarium (Fig. 2) for 3-4 hours in laboratory condition. For the final experiment, only the healthy and mobile fishes were selected. During the experiment, the seeds were crushed into fine powder firstly in a mortar with a pestle and then grinded in an electric blender. 382 Sultana et al.
Fig. 2: Acclimatization of Heteropneustes fossilis in the laboratory.
The experimental seed powder was sieved through a sieve of 0.0025 cm² mesh size and was extracted with distilled water, 50% ethyl alcohol, methanol and acetone solvents separately. For experimental purposes the required amount of seed powder was weighed and mixed with relevant amount of the solvent in a glass Stoppard conical flask and kept on a magnetic stirrer for 3-4 hours. After extraction, the resultant liquid was filtered through filter paper (Whatman No. 1) and the different test doses were prepared from this stock solution. Desired concentrations of different test solutions were obtained by appropriate dilution of the stock solution (APHA 1995) which was prepared on the day of the experiment. Procedure of bioassays: Experiments (Fig. 3) were done to determine 1- 99% mortality of the liquid extracts obtained from the dry seed powder of J. carcus, H. wightianus and A. moluccana against the test fish H. fossilis. A set of glass aquarium (30 x 23 x 23 cm3) were used for the bioassays. Each of the aquarium contained 5L of tap water, toxicant and five test fish. The different proportions of seed extracts were added to obtain the required concentrations, so as to determine the LC50 values of the seed extracts for the experimental fish. Five concentrations of each extract in triplicates were used in the final experiments after preliminary screening. A set of five healthy and active fishes were then released in each concentration randomly. The test fishes were kept in exposure to the toxicated water for 24±2 hours at room temperature (29±2°C) and in diffused light. A control set was maintained in each experiment which contained 5L of water and same number of fish. Behavioral pattern of control fishes were also observed and noted at that time for comparison with the affected fishes by the toxicants. Mortality of fishes was recorded only of those fishes which were killed within 24 hours after exposure to the various concentrations of the toxicants. Toxicological effect three indigenous plant seed extracts 383
Fig. 3: Experimental set of test of seed extracts.
Statistical analysis: To determine the LC50 values of the dry seed extracts on the experimental fishes, probit analysis was done. The values of empirical probit, working probit and weighting probit were taken from the tables of Finney (1971). The regression equation was calculated from empirical probit, working probit and weighting probit. Values of LC50 with 95% confidence intervals were analyzed in a computer based probit analysis programme. The value of chi- square (χ2) test was determined (Fisher and Yates 1963) and compared with tables of the statistics for (n-1) degrees of freedom at 1% and 5% level of significance. Analysis of variance of percentage mortality of fishes was made to estimate the variation among treatments and replicates at 0.01 and 0.05 levels of significance. Toxicity values or relative potency values were calculated on the basis of potency which is a reciprocal of the highest concentration. The relative potency of the toxicants was obtained by taking the highest LC50 values as unit and dividing the highest LC50 value of a toxicant with the respective LC50 value of other toxicants.
RESULTS AND DISCUSSION Effect of the three seed extracts on behavior of H. fossilis: The differences in the behavioral changes with the seed extracts of J. carcus, H. wightianus and A. moluccana were observed in H. fossilis. With the acetone extract the affected fishes showed restlessness with erratic swimming from the very beginning with frequent gulping of air. Reddish colour was seen in the opercular and gill region in the dead fishes, indicating internal hemorrhage in the gills, as the acetone extract was the highly toxic extract. With the methanol extract huge slime secretion was seen. Most of the dead fishes floated upwards, lying parallel to the water surface of the aquaria. The characteristics seen in the fishes with other less toxic extracts were mainly that they showed erratic and abnormal behavior after a long time. The dead fishes floated at different angles as 45°, 60° and 90° or lying at the bottom of the aquarium with large and round mouth opening, damaged barbles, straight opercular fins, pale 384 Sultana et al. skin and bulging eyes (Fig. 4). Hence with respect to behavior the toxicity of the four extracts could be ranked as: Acetone Methanol 50% ethyl alcohol distilled water. According to toxicity of seeds, J. carcus seed extracts were the most toxic extract showing abnormalities, such as irregular swimming, moving up and down throughout the water, settling down at the corner or bottom of the aquaria, slowly gulping for air, inactivity, slow upward movement, becoming balance less, inactive and finally died within 24 hours.
Fig. 4: Behavioral responses of H. fossilis treated with experimental plant seed extracts.
Effects of extracts on mortality: The dose ranges, mortality ranges, regression equations of the slope lines, chi-square and ANOVA values and their significance at 0.01 and 0.05 levels, and LC50 values of the three experimental plant seed extracts on H. fossilis are given in Table 1. Doses with J. carcus seed extracts ranged from 3-85 ppm, with H. wightianus seed extracts from 50-2000 ppm and with A. moluccana seed extracts from 550-2500 ppm. Mortality of the experimental fishes ranged from 6.67-93.33%. Chi-square values at 0.01 and 0.05 levels of significance showed that most of the extracts had insignificant values except 50% ethyl alcohol extract of J. carcus and A. moluccana seeds, and methanol extracts of H. wightianus and A. moluccana seeds. ANOVA test showed Toxicological effect three indigenous plant seed extracts 385 that the treatment values were significant with all the extracts at 0.01 and 0.05 levels excepting methanol extract of H. wightianus seed at 0.01level of significance. The replicate values were insignificant with all the extracts at 0.01 and 0.05 levels. The LC50 values showed that amongst all the experimental seed extracts, acetone extract of J. carcus seeds was the most toxic extract and distilled water extract of A. moluccana seeds was the least toxic extract. From
Table 2 it is observed that the lowest LC50 value was found with acetone extract of J. carcus seeds and it was the most toxic extract having the LC50 value 6. 865 ppm and high relative potency value 217.05. The highest LC50 value was observed with distilled water extract of A. moluccana seeds. Its LC50 value was 1490.056 ppm and relative potency value was 1.00. Hence, it was the least toxic extract. Distilled water, 50% ethyl alcohol and methanol extracts of J. carcus seeds with LC50 values of 60.084 ppm, 23.627 ppm, 16.015 ppm and with good relative potency values of 24.80, 63.07 and 93.04 respectively were also highly toxic; acetone and methanol extracts of H. wightianus seeds with LC50 values of 291.749 ppm and 439.860 ppm and with relative potency values 5.11 and 3.39 respectively were toxic; fifty percent ethyl alcohol extract of H. wightianus, and acetone extract of A. moluccana seeds with LC50 values of 895.912 ppm, and 906.998 ppm with relative potency values of 1.66 and 1.64 were medium toxic extracts; methanol extract of A. molluccana seeds with LC50 of 1018.185 and relative potency of 1.46 was fairly toxic; fifty percent ethyl alcohol extract of A. moluccana seeds and distilled water extract of H. wightianus seeds with LC50 of 1274.335 ppm, and 1396.726 ppm and with relative potency values of 1.17 and 1.07 were less toxic extracts. On the basis of their relative potency values, i.e. toxicities, the relative position of the extracts for H. fossilis were as follows: Acetone extract of J. carcus seeds > Methanol extract of J. carcus seeds > 50% ethyl alcohol extract of J. carcus seeds > Distilled water extract of J. carcus seeds > Acetone extract of H. wightianus seeds > Acetone extract of A. moluccana seeds > Methanol extract of H. wightianus seeds > Methanol extract of A. moluccana seeds > 50% ethyl alcohol extract of H. wightianus seeds > 50% ethyl alcohol extract of A. moluccana seeds > Distilled water extract of H. wightianus seeds > Distilled water extract of A. moluccana seeds. There are a good number of herbs, shrubs and indigenous plants in Bangladesh which have piscicidal property. The present investigation on indigenous plants is to explore the piscicidal activities of seeds of three indigenous plants Jatropha carcus (Bengali Jamal gota), Hydnocarpus wightianus (Bengali Chalmogra) and Aleurites moluccana (Bengali Kukui nut)
386 Sultana et al.
Table 2: LC50 and Relative potency values with categories of distilled water, 50% ethyl alcohol, methanol and acetone extracts of J. curcas, H. wightianus and A. moluccana seeds on H. fossilis
Plant seed Extract LC50 (ppm) Relative potency Category Distilled water 60.084 24.80 Highly toxic Jatropha 50% ethyl alcohol 23.627 63.07 Highly toxic carcus Methanol 16.015 93.04 Highly toxic Acetone 6.865 217.05 Most toxic Distilled water 1396.726 1.07 Less toxic Hydnocarpus 50% ethyl alcohol 895.912 1.66 Medium toxic wightianus Methanol 439.860 3.39 Toxic Acetone 291.749 5.11 Toxic Distilled water 1490.056 1.00 Least toxic Aleurites 50% ethyl alcohol 1274.335 1.17 Less toxic moluccana Methanol 1018.185 1.46 Fairly toxic Acetone 906.998 1.64 Medium toxic with different solvents such as distilled water, 50% ethyl alcohol, methanol and acetone extracts were determined against predatory fish Heteropneustes fossilis as the test fish. All the extracts were more or less toxic against the dose concentrations. The solubilizing capacity of the solvents for the three experimental seeds on H. fossilis on the basis of experimental analysis could be ranked in the order: Acetone > Methanol > 50% ethyl alcohol > Distilled water extracts. On the basis of ranking, it can be explained as that the plant components were mostly soluble in acetone, followed by methanol, then 50% ethyl alcohol and less soluble in distilled water which agrees with strong solubility in absolute ethyl alcohol, Toxicological effect three indigenous plant seed extracts 387 slight solubility in 50% ethyl alcohol and almost insoluble in distilled water of finding of Ameen et al. (1983). All the seed extracts showed to be more or less toxic with variations of concentrations. Various reactions were shown by the test fishes with special regards to their movement. Mortality of fishes was dependant on concentrations in all the extracts with all the three seed extracts. The fishes showed various abnormalities in their behavior within about 24 hours exposure to the concentrations. They displayed restlessness, frequent surfacing, irregular swimming, jerky movements, gulping air and extensions of fins and barbels. Erratic movements were also shown by the affected fish with gradual loss of balance and slime secretion, followed by death. Ultimately they died with large and wide mouth opening and with straight and spreaded fins. Faded colour and damaged gills, fins and barbels were also observed in the dead fishes. Some dead fishes were found floating at the surface of the aquarium with ventral side upward and some other fishes were floating at different angles. In some cases blood was seen around from the mouth of the fish. These findings are in agreement with the findings of Latifa et al. (1997), Latifa and Begum (1993) and Nasiruddin et al. (2006, 2011, 2012a, b, 2014). With respect to behavior in H. fossilis, toxicity of the four extracts was in the order: Acetone > Methanol > 50% ethyl alcohol > Distilled water extracts. With respect to seeds, the toxicity of the three seeds was in the order: J. carcus > H. wightianus > A. moluccana seeds.
The LC50 values also showed the trend of toxicity in the three seed extracts being ranked in the order: Acetone > Methanol > 50% ethyl alcohol > Distilled water. From the present study it was observed that amongst all the seed extracts the highest toxic extract was acetone extract of J. carcus seeds with concentration ranges between 3-15 ppm and with LC50 value 6.865 ppm whereas the least toxic extract was the distilled water extract of A. moluccana seeds at concentration ranges 750-2500 ppm with LC50 value 1490.056 ppm.
On the basis of LC50 values of distilled water extracts, the LC50 value of J. carcus seeds (60.084 ppm) showed close similarity with LC50value of Albizia procera seeds (74.79 ppm) of Chowdhury et al. (2014), whilst LC50 values of H. wightianus seeds (1396.726 ppm ) and A. moluccana seeds (1490.056 ppm) showed similarity with LC50 value of Phyllanthus emblica seeds (1488.60 ppm) of Nasiruddin et al. (2014).
On the basis of LC50 values of 50% ethyl alcohol extracts, the LC50 value of
J. carcus seeds (23.627 ppm) was very close to the LC50 of Albizzia procera seeds
(24.81 ppm) of Nasiruddin et al. (1998); LC50 value of H. wightianus seeds
(895.912 ppm) was almost similar to LC50 value of P. emblica seeds (890.98 ppm) of Nasiruddin et al. (2014); and LC50 values of A. moluccana seeds (1274.335 388 Sultana et al.
ppm) was more or less close to the LC50 value of Terminalia bellerica seeds (1171.07 ppm) of Nasiruddin et al. (2014), Datura innoxia seeds (1337.73 ppm) and Acacia catechue seeds (1341.99 ppm) of Nasiruddin et al. (2014).
On the basis of LC50 values of methanol extracts, the LC50 value of H. wightianus seeds (439.860 ppm) was close to that of P. emblica seed extract
(489.37 ppm) of Nasiruddin et al. (2014) and LC50 value of A. moluccana seeds
(906.998 ppm) was somewhat near to LC50 value of T. bellerica seeds (777.43 ppm) of Nasiruddin et al. (2014). Whilst LC50 value of acetone extract of H. wightianus seeds (291.749 ppm) was somewhat close to the LC50 value of acetone extract of Terminalia chebula (247.749 ppm) seeds of Nasiruddin et al. (2014). Chi-square values at 0.05 and 0.01 levels of significance showed that most of the extracts had insignificant values except 50% ethyl alcohol extract of J. carcus and A. moluccana seeds, and methanol extracts of H. wightianus and A. moluccana seeds which indicated a good relationship between the observed and expected mortalities. ANOVA test showed that the treatment values were significant with all the extracts at 0.01 and 0.05 levels except methanol extract of H. wightianus seeds at 0.0 level of significance. The replicate values were insignificant with all the seed extracts at 0.01 and 0.05 levels, hence, giving an indication of a good relationship between the concentrations applied and mortalities obtained. It was apparent from the present study that the potentiality of different extracts like distilled water, 50% ethyl alcohol, methanol and acetone extracts of J. carcus, H. wightianus and A. moluccana seeds appeared to be promising in different concentration levels, and as such, they might be effective for controlling and preventing the undesirable or unwanted predatory fish species in different ponds such as nursery, rearing and stocking ponds of commercially valuable fish species. It is revealed that, various information about the efficiency of the plant toxins can be obtained from the laboratory based toxicity studies of crude dry powders. Therefore, for developing the effects of these plant seed powder on ecological systems, toxicity data of the present study is essential.
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(Manuscript received on 14 February, 2020 revised on 6 October, 2020)